Site‐Selective Coordination Assembly of Dynamic Metal‐Phenolic Networks

Abstract Coordination states of metal‐organic materials are known to dictate their physicochemical properties and applications in various fields. However, understanding and controlling coordination sites in metal‐organic systems is challenging. Herein, we report the synthesis of site‐selective coordinated metal‐phenolic networks (MPNs) using flavonoids as coordination modulators. The site‐selective coordination was systematically investigated experimentally and computationally using ligands with one, two, and multiple different coordination sites. Tuning the multimodal Fe coordination with catechol, carbonyl, and hydroxyl groups within the MPNs enabled the facile engineering of diverse physicochemical properties including size, selective permeability (20–2000 kDa), and pH‐dependent degradability. This study expands our understanding of metal‐phenolic chemistry and provides new routes for the rational design of structurally tailorable coordination‐based materials.


Section S1.2 Characterization
UV-vis absorption spectra were recorded on a Specord 250 Plus spectrophotometer (Analytik Jena AG). Fourier transform infrared (FTIR) spectroscopy analysis was conducted on a Tensor-II-FTIR spectrometer. Differential interference contrast (DIC) microscopy images of capsules were taken with an inverted Olympus IX71 microscope. Scanning electron microscopy (SEM) images were obtained using an FEI Quanta 200 field-emission scanning electron microscope, operating at an accelerating voltage of 10 kV. For the SEM experiments, dried samples were coated with Au by using K575X Turbo Sputter Coater. Transmission electron microscopy (TEM) and energy-dispersive X-ray mapping analysis of capsules were performed on an FEI Tecnai TF20 instrument (USA) at an operating voltage of 200 kV. Atomic force microscopy (AFM) experiments were conducted using a JPK NanoWizard II BioAFM instrument. Confocal laser scanning microscopy (CLSM) images were taken with a Nikon A1R+ laser scanning confocal microscope (Nikon Corporation, Japan). ζ-Potential measurements were performed using a Malvern Zetasizer Nano ZS instrument (Malvern Instrument, UK). The metal content in the capsules was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using an ICP Varian 710-ES instrument. QUE, Fe ion, EDTA, and Fe II -QUE metal-phenolic network (MPN) components after degradation of the MPN QUE capsules by EDTA were purified using high-performance liquid chromatography (HPLC) equipped with a UV detector (Shimadzu, Kyoto, Japan). HPLC analysis was performed on a C18 column (4.6 mm internal diameter (ID) using gradient mode with water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The flow rates, operating temperature, and UV wavelength for detection were set at 1 mL min −1 , 35 °C, and 254 nm, respectively.The chemical structures of free QUE and QUE after MPN assembly procedure were determined by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy on a 400 MHz NMR spectrometer (JEOL Ltd., Akishima, Tokyo, Japan).

Section S1.3 Quantum Mechanical (QM) Methodology
All density functional theory (DFT) and wavefunction calculations were performed in ORCA 5.0.1. [1] Geometry optimizations were performed using the B97M-V functional [2] and def2-TZVP basis set, [3] using the resolution of the identity (RIJ) approximation and a def2/J auxiliary basis set. [4] These optimizations were performed directly in a conductor-like polarizable continuum model (CPCM) solvent field, [5] with water as the solvent. Improved energies were calculated with DLPNO-CCSD(T) [6] and the cc-pVTZ basis set, [7] using the RIJCOSX approximation and the cc-pVTZ/C and def2/J auxiliary basis sets. [4,8] These DLPNO-CCSD(T) single point calculations were also performed in a CPCM solvent field. For calculations involving anionic Fe(III) complexes (and their conjugate acids), DLPNO-CCSD(T)/aug-cc-pVTZ calculations were performed, using an aug-cc-pVTZ/C auxiliary basis set. Due to linear dependency issues, the RIJCOSX approximation was not used for these calculations. All DFT and DLPNO-CCSD(T) calculations were performed using 'Tight' Self-Consistent Field (SCF) cut-offs.
Having optimized the structures, (numerical) frequencies were calculated in the CPCM solution to confirm structures were true minima, possessing no imaginary frequencies. Solution-phase Gibbs free energies were then calculated using the "direct method". [9] For systems that undergo large geometry changes upon solvation, the direct method affords energetics that are superior to those obtained using (gas phase to solution) thermocycles. [9] Given the high partial charges on atoms within these metal complexes, significant geometry changes upon solvation (in water) would be anticipated. The default standard state used within ORCA 5.0.1. for entropic components (even in a CPCM solvent field) is based on the statistical mechanics for an ideal gas (evaluated at 1 atm of pressure and 25 °C). Thus, appropriate standard state corrections were applied to ensure all binding energies and pK a values were calculated at a standard state for solutes in solution (of 1 mol L −1 and 25 °C). This correction takes the following form.
Here Δm is the change in moles upon reaction, R is the ideal gas constant (8.3145 J mol -1 K -1 ), T is the temperature of interest at which the Gibbs free energy is also evaluated (typically 298.15 K) and P is pressure (101.325 kPa = 1 atm).
For reactions that either generate or consume water (i.e., reactions where water is a product or reagent), a further state correction is required. The standard state for any liquid is the pure substance at 1 atm of pressure, so the standard state of liquid water should be [H 2 O] = 55.5 mol L −1 (rather than 1 mol L −1 for an aqueous solute). Gibbs free energies for reactions involving water as a reagent must be further corrected by adding the following additional term: Here n is the number of moles of water acting as a reagent. Conversely, for reactions that generate water as a product, Gibbs free energies are corrected by the following term: Here n is the number of moles of water produced. We should emphasize these standard state corrections are only required for reaction energies calculated via QM approaches.
For QUE and DHF systems, both syn-and anti-conformations of the free ligand and respective Fe II and Fe III complexes were considered for all protonation states. Theoretically, any of the unique aqua ligands can undergo deprotonation, with this process forming distinct coordination isomers (e.g., with OH groups in either axial or equatorial positions). Thus, deprotonation of all unique aqua ligands was considered to identify the most energetically favorable coordination geometry for a given overall protonation state. Similarly, for DHF and QUE complexes (with Site B binding), deprotonation of the Fe-bound para-and meta-OH group was considered, as these processes lead to different coordination isomers. In all cases, reported energetics are based on the lowest energy conformation identified. All Fe II and Fe III complexes were modeled in their respective high-spin (quintet and sextet) configurations as test calculations indicated these were significantly more stable than possible low-spin states. Molecular graphics were rendered with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.37. [10] Section S1.

Calculation of pKa Values and Binding Constants
Within standard continuum solvation models, solvation energies are profoundly influenced by the overall charge of the metal-ligand (ML) complex as well as the coordination number (CN) and oxidation/spin state of the metal ion. [11] Absolute predictions of pK a values for ML complexes are prone to significant solvation errors because deprotonation decreases the overall charge of the complex and often decreases the CN of the metal ion. [11] However, solvation errors are largely systematic and can be offset by considering concurrent protonation of a (structurally similar) reference conjugate base. [12]  As these hydrolysis reactions (Rxns 1-6) are important for describing the acid/base characteristics of iron species in aqueous environments, the reference pK a values are known experimentally with relatively high precision.
Speciation of these Fe III aqua complexes in different protonation states has been determined in previous work (see Ref [13] ). The corresponding reference experimental pK a values for these Fe III complexes were taken from Ref [13] . However, we should note that there is some experimental variability in these values, particularly for Rxn 5 and Rxn 6. For instance, Ref [14] reports pK a values for Rxn 5 that are around 2 units higher (8.54) and values for Rxn 6 that are around 2 units lower (7.41) than the corresponding reference experimental pK a values from Ref [13] . Although the choice of reference values does not impact the site selectivity of the respective complexes directly (see Table S9 below), it does impact the pH range where these complexes predominate (see Figure S1). These reference values are also dependent on ionic strength (I). [14] The reported pK a values for Rxns 3-6 are 2.9, 3.9, 6.5, and 8.6, respectively, at I = 0.5 mol L −1 , and 2.7, 4.3, 5.5, and 8.1, respectively, at I = 1.0 mol L −1 . [15] Formally, the QM calculations are performed at I = 0 mol L -1 , thus the reference pK a values taken at I = 0 mol L −1 were used for Rxns 1-6. However, the effect of non-zero ionic strength could be roughly approximated by using reference pK a values at I = 0.5 and 1.0 mol L −1 (see Figure S2).  [16] ), the precise structure and speciation of [Fe II (H 2 O) n (OH) 2 ] 0 is more speculative. Experimental identification of this species is difficult because of the poor solubility of Fe II species at high pH and their instability toward aerobic oxidation. [17] Figure S3).
Accounting for the necessary standard state corrections (see Eqs 1-3 above), the trigonal-bipyramidal [Fe II (H 2 O) 3 (OH) 2 ] 0 complex was found to be the most stable and thus was assumed to be the dominant species in Rxn 2. The corresponding experimental pK a values for these Fe II aqua species were taken from Ref [17] . Rxns 7 and 8 compensate for the decrease in the overall charge of a given ML complex (upon deprotonation) via a corresponding increase to the overall charge of a reference metal-aqua complex (through protonation). These metal-aqua complexes are chosen so that the reference acid has the same overall charge as the metal-ligand complex in its protonated form. These proton-transfer reactions also conserve the oxidation states and total CN of the metal ions.
Relative pK a values for the ML complexes of interest, with respect to the corresponding metal-aqua complexes, can be determined via the Gibbs free energies for these isodesmic proton-transfer (PT) reactions (ΔG PT ): Whereas most of these proton-transfer reactions are isodesmic, those involving reference Rxn 6 are not because water is required as a reagent to balance the reaction. For instance, for a model diprotic ligand (LH 2 ), the proton-transfer reaction is: For reactions of the form of Rxn 9, the QM-calculated Gibbs free energies must be corrected according to Eq. 2 to account for the consumption of water. The energetics for Rxns 13 and 14 can be determined using similar Hess's Law thermocycles and the pKa values of the free ligands: Although many of the pKa values of the free flavonoid ligands are known experimentally, they can also be calculated using a similar relative pKa approach (as described above). Although binding energies could be further corrected to account for the impact of metalaqua complex hydrolysis reactions and free ligand ionization, neither of these processes impacts on relative site selectivity (though absolute effective binding energies are affected). Given the focus was on relative predictions of site selectivity, neither ligand ionization nor metal-aqua complex hydrolysis was explicitly considered in this work.
Section S1.5 Determination of Coordination Sites UV-vis spectrophotometry was used to determine the coordination sites of Fe II -flavonoid complexes in aqueous solution. The samples were prepared as follows: solutions of free flavonoids dissolved in methanol to achieve a concentration of 0.25 mM were prepared. The Fe II -QUE complexes were obtained by mixing 0.5 mM FeCl 2 ·4H 2 O in water with 0.25 mM QUE in methanol, and the pH of the complex solution was adjusted to within a range of 1-12 by adding 1 mM NaOH or 1 mM HCl solution. To obtain the other Fe II -flavonoid complexes, 0.25 mM 3HF, CHR, or DHF was mixed with 0.5 mM FeCl 2 ·4H 2 O, and the solution of the complex was adjusted to pH 4, 7, or 9. FTIR spectroscopy was used to determine the coordination between Fe II and flavonoids in the solid state. The samples were prepared as follows: 500 μL of FeCl 2 ·4H 2 O (10 mg mL −1 in water) and 500 μL of QUE (5 mg mL −1 in methanol) were added and vortexed for 10 s, and the pH of the mixed solution was adjusted by adding 1000 μL of 100 mM MOPS (pH 4, 7, or 9). The mixture was kept still for 2 h. The free complexes were removed by centrifugation (2000 g, 1 min) and the sediments were washed three times with Milli-Q water. The Fe II -QUE complexes were freeze-dried before the FTIR spectroscopy measurements. Other Fe II -flavonoid complexes were prepared in the same manner as described for the Fe II -QUE complexes. The Gaussian function fitting approach was used to perform curve fitting.

Section S1.6 Fabrication of MPN Capsules from Particle Templates
All flavonoids and metal solutions were prepared freshly for immediate use. The standard protocol used for capsule preparation was as follows: 30 μL of PS particles was washed twice with Milli-Q water (2000 g, 1 min) and suspended in 365 μL of water. Then 75 μL of methanol, 13.2 μL of FeCl 2 ·4H 2 O (10 mg mL −1 in water), and 20 μL of QUE (5 mg mL −1 in methanol) were added successively to the PS dispersion at room temperature (25 °C) followed by brief vortexing and sonication. The pH of the mixed dispersion was adjusted by adding 500 μL of 100 mM MOPS (pH 4, 7, or 9) and the suspension was vortexed for 60 s. The mixture was allowed to sit undisturbed for 2 h to achieve sufficient film formation and adherence. Non-coating complexes were removed by centrifugation (2000 g, 1 min) and the pellet was washed three times with Milli-Q water. To obtain MPN capsules, 1000 μL of THF was added to dilute the suspension and the particles were incubated with THF for at least 3 h. Then the pellets were washed four times with THF (2000 g, 3 min), and the resulting hollow capsules were resuspended in 300 μL of water for characterization. The same protocol was followed for the fabrication of MPN capsules using other particle templates.
For the preparation of MPN coatings with other flavonoids, 40 μL of 2.5 mg mL −1 3HF or LUT, 100 μL of 1 mg mL −1 CHR, or 50 μL of 2 mg mL −1 DHF or FIS was used, and the same fabrication process was applied. For fabricating MPN coatings at pH 4 using different metal ions and ligands, 15 μL of 5 mg mL −1 ZrCl 4 or 11 μL of 15 mg mL −1 AlCl 3 ·6H 2 O and 40 μL of 2 mg mL −1 DHNQ, 31 μL of 2 mg mL −1 DHAQ, or 56 μL of 10 mg mL −1 TA were used, and the same fabrication process was applied.

Section S1.10 Permeability Experiments
To test capsule permeability, 200 μL of an MPN-coated PS suspension was added to the modified microscopy glass substrate and then the substrate was kept still for 10 min. The glass substrate was then immersed in THF to remove the PS templates and rinsed with Milli-Q water. Then, 200 μL of FITC-dextran (1 mg mL −1 in solution at the desired pH; M w = 20, 70, 250, 500, 2000 kDa) was added to the capsule area on the glass substrate and incubated for 10 min. The capsules (n = 100) were examined by CLSM. Capsules with dark interiors were regarded as impermeable, whereas those with interiors of the same fluorescence intensity as the outer environment were considered to be permeable.

Section S1.11 Dynamic Size Measurements
For the dynamic size measurements, 10 μL of an MPN-coated PS suspension was added to the modified microscopy glass substrate and then the substrate was kept still for 5 min. Then, 200 μL of 1,4-dioxane was added in situ to remove the PS templates, and excess 1,4-dioxane was aspirated. Subsequently, 200 μL of 100 mM MOPS (pH 4, 7, or 9) was added to the capsule area on the glass substrate and incubated for 10 min. For measuring the MPN capsule size under the above solutions, the diameter of the capsules (n = 20) was measured by DIC microscopy.

Section S1.12 Cytotoxicity Assay of MPN Capsules
The XTT-based in vitro cytotoxicity assay was performed to assess cell toxicity of the MPN capsules. XTT was dissolved in complete DMEM (with 10% FBS) to form 0.2 mg mL −1 solution, and phenazine methosulfate (PMS) was dissolved in DPBS to form 1 mM solution.
The XTT reagent was activated by mixing with PMS solution at a volume ratio of 400:1. PC-3 cells or A549 cells were seeded on a 96well plate at a cell density of 2 × 10 4 cells per well. After incubation with the capsules at different drug dosages for 48 h, the media in the 96-well plate was aspirated and replaced with 100 μL of fresh activated XTT media. The cells were further incubated for 4 h and the absorbance at 475 nm was measured relative to non-treated cells.

Section S1.13 Minimum Information Reporting in Bio-Nano Experimental Literature (MIRIBEL)
The studies conducted herein, including material characterization, biological characterization, and experimental details, conform to the MIRIBEL reporting standard for bio-nano research, [18] and we include a companion checklist of these components herein. Figure S1. Schematic of the coordination-driven assembly and pH-dependent disassembly between organic ligands with multiple chelation sites and metal ions. Figure S2. Comparison of the predicted QUE site selectivity for Fe III as a function of pH using reference hydrolysis data from Ref [13] (solid lines) and Ref [14] (dashed lines). Figure S3. Comparison of the predicted QUE site selectivity for Fe III as a function of pH using reference hydrolysis data from Ref [15] taken at I = 1.0 (solid lines) and 0.5 mol L −1 (dashed lines).                                      The "TOP guidelines": e.g., Science 352 (2016) 1147; http://doi.org/10.1126/science.aag2359 Similar to many of the efforts listed above, the parameters included in this checklist are not intended to be definitive requirements; instead they are intended as 'points to be considered', with authors themselves deciding which parameters are-and which are notappropriate for their specific study.

Section S3. Supplementary Tables
This document is intended to be a living document, which we propose is revisited and amended annually by interested members of the community, who are encouraged to contact the authors of this document. Parts of this document were developed at the annual International Nanomedicine Conference in Sydney, Australia: http://www.oznanomed.org/, which will continue to act as a venue for their review and development, and interested members of the community are encouraged to attend.
After filling out the following pages, this checklist document can be attached as a "Supporting Information" document during submission of a manuscript to inform Editors and Reviewers (and eventually readers) that all points of MIRIBEL have been considered. *For in vitro experiments (e.g., cell culture), ex vivo experiments (e.g., in blood samples), and in vivo experiments (e.g., animal models). The questions above that are appropriate depend on the type of experiment conducted.